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Dev Biol
2004 Apr 15;2682:280-94. doi: 10.1016/j.ydbio.2003.12.035.
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ALK4 functions as a receptor for multiple TGF beta-related ligands to regulate left-right axis determination and mesoderm induction in Xenopus.
Chen Y
,
Mironova E
,
Whitaker LL
,
Edwards L
,
Yost HJ
,
Ramsdell AF
.
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In Xenopus, several TGF betas, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left-right (LR) defects when ectopically targeted to rightmesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGF betas. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGF beta receptor(s) are involved in mediating left-side TGF beta signaling. We report here that similar to effects of ectopic TGF betas, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGF beta(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGF betas, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGF betas is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGF betas to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.
Fig. 3. Perturbation of Pitx2 expression by ALK4. Xenopus blastulae were injected with CA-ALK4 RNA in left (L3) or right (R3) cell lineages and control and experimental embryos were processed for whole-mount in situ hybridization to detect Pitx2 expression in the lateral plate mesoderm (ventral view, arrowheads). The percentages of embryos exhibiting normal (left side) or abnormal (bilateral or right side only) expression patterns are indicated.
Fig. 2. Left–right axis defects elicited by CA-ALK4 expression. Control and experimental embryos were scored for LR phenotype at day 5 postinjection of CA-ALK4 RNA. The orientation of embryos shown is a ventral view, with anterior at the top. Control embryos exhibit situs solitus, as defined by rightward heart looping (arrowhead) plus counterclockwise coiling and leftward rotation of the gut (A). In contrast, embryos with right-side CA-ALK4 expression exhibit situs inversus, as defined by complete reversal of the heart and gut (B), or situs ambiguus, as defined by reversed heart looping plus normal gut orientation (C) or normal heart looping plus reversed gut orientation (D). The vegetal cell lineages that were targeted for left-side (L3) or right-side (R3) RNA expression are indicated in an illustration of a 16-cell embryo (E, animal pole view). As a control for ALK4 antisense experiments (F), protein levels were examined by Western blot analysis in embryos injected with epitope-tagged ALK4 in the absence and presence of ALK4 morpholino (MO). The ALK4 MO is effective in reducing expression of ectopic ALK4 by approximately 30% when densitometry readings are averaged over the course of three independent experiments. By comparison, levels of a control protein, pinched, are not decreased by co-expressed ALK4 MO.
Fig. 5. Rescue of Xnr1-induced dorsoanterior defects by DN-ALK4. Right cell lineages of Xenopus blastulae were injected with RNA or cDNA encoding TGFβs in the absence or presence of DN-ALK4 RNA as indicated in Methods. Embryos were imaged at 3–5 days following injections and incubation at 16°C. Expression of AVg RNA alone (A) results in some embryos that are ventralized with excess endoderm formation and others that exhibit secondary axis formation (arrows). DN-ALK4 co-expression (B) blocks both types of defects. Expression of Xnr1 pCSKA alone (C) results in embryos that are ventralized as characterized by shortened primary axes and absent eye development on the injected side of the embryo. In addition, many embryos exhibit tail defects resulting from incomplete blastopore closure (arrowheads). DN-ALK4 co-expression blocks the ventralizing properties of Xnr1 pCSKA (D), although minor tail defects still occur in some embryos (arrowheads). Expression of Xnr1 pXeX in the absence (E) or presence (F) of DN-ALK4 results in embryos that have normal primary axis formation and no discernible tail defects. Expression of Xnr1 RNA alone (G) results in embryos that are hyperdorsalized and DN-ALK4 co-expression (H) blocks this effect, although some embryos still exhibit posteriortail truncation (arrowheads). Expression of derriere RNA alone (I) results in embryos that are extremely ventralized and DN-ALK4 co-expression (J) blocks this effect. See Table 3 and text for details.
